This is an R01 competing continuation application to investigate the mechanism of action of nitric-oxide donating aspirin (NO-ASA) against colon cancer. NO-ASA consists of aspirin and a NO-releasing moiety linked to it via a chemical spacer. In the last few years we have pursued the application of NO-ASA for the prevention of colon cancer as a safer and more potent alternative to conventional aspirin. We have observed that NO-ASA induces a series of changes in the colon cell, concerning both cell kinetics and cell signaling. Importantly, NO-ASA a) increases the levels of several reactive oxygen and nitrogen species (collectively referred to as ROS); b) activates MAPK signaling, which mediates its cell growth inhibitory effect; and c) inhibits cell growth. Our hypothesis is that NO-ASA prevents colon cancer efficiently and safely, primarily by increasing the levels of several ROS in the colonocyte, which activate MAPK signaling leading to inhibition of cell growth. Elucidating this biochemical sequence will help us optimize a NO-ASA-centered strategy for colon cancer prevention. To evaluate this hypothesis and delineate the steps involved in these effects, we propose to: 1) Identify and assay ROS generated by NO-ASA and assess the mechanism of their production. 2) Determine the mechanism by which NO-ASA-induced ROS inhibit colon cancer cell growth through the JNK and p38 MAPK pathways. 3) Determine the relevance to chemoprevention of these effects of NO-ASA both in animal models of colon cancer and in humans participating in a clinical trial evaluating NO-ASA as a chemopreventive agent. Our preliminary data support these aims and we expect that the proposed studies will help us develop an efficient and safe approach to colon cancer prevention. [unreadable] [unreadable] [unreadable]